High throughput methods for creating and analyzing chemical and biochemical diversity play a vital role in technologies including drug discovery and development. Specific applications of high throughput methods include drug discovery, optimization of reaction conditions (e.g., conditions suitable for protein crystallization), genomics, proteomics, genotyping, polymorphism analysis, examination of RNA expression profiles in cells or tissues, sequencing by hybridization, and recombinant enzyme discovery.
Rapid, high throughput methods for synthesizing (e.g., using combinatorial chemistry methods) and screening large numbers of these compounds for biological and physicochemical properties are desired, for example, to increase the speed of discovery and optimization of drug leads.
Similarly, due in part to the large amount of sequence data from the human genome project, efforts are underway to rapidly obtain x-ray crystallography data for the protein products of many newly discovered genes. One of the rate limiting steps in this process is the search for appropriate solution conditions (e.g., pH, salt concentration) to cause protein crystallization. There is also a need to determine the function of each of the newly discovered genes (i.e., “functional genomics”) and to map protein-protein interactions (i.e., “proteomics”). Given the large number of human genes, protein modifications, and protein binding partners, higher throughput methods are desired.
Another advance in biotechnology is the creation of surfaces with high-density arrays of biopolymers such as oligonucleotides or peptides. High-density oligonucleotide arrays are used, for example, in genotyping, polymorphism analysis, examination of RNA expression profiles in cells or tissues, and hybridization-based sequencing methods as described, for example, in U.S. Pat. Nos. 5,492,806, 5,525,464, and 5,667,972 to Hyseq, Inc. Arrays containing a greater number of probes than currently provided are desirable.
The process of discovering and improving recombinant enzymes for industrial or consumer use has emerged as an important economic activity in recent years. A desire to discover very rare, activity-improving mutations has further stimulated the search for higher throughput screening methods. Such methods often require screening 100,000 to 1,000,000 members of a genetic library in parallel, and then rapidly detecting and isolating promising members for further analysis and optimization.
One of the challenges in the development of high throughput methods is that conventional liquid handling techniques such as pipetting, piezoelectric droplet dispensing, split pin dispensing, and microspritzing are generally unsuitable for rapidly loading or transferring liquids to or from plates of high density (e.g., plates having more than about 384 wells). For example, these techniques can cause substantial splashing, resulting, for example, in contamination of neighboring wells and loss of sample volume. Also, as the number of wells increases, the time necessary to reformat compounds from the previous generation of plates to the higher density plates generally increases, thus limiting the utility of higher density plates. Evaporation can also be problematic with times greater than a few seconds. Moreover, entrapped air bubbles can result in inconsistencies in the loading of small fluid volumes (e.g., less than about one microliter).
Significant bottlenecks in high throughput screening efforts include library storage, handling, and shipping. As the number of compounds in a library increases, the number of 96- or 384-well plates, and the total volume needed to store the libraries, also increases. For compounds that are stored in frozen solvent such as DMSO or water, thawing, dispensing, and refreezing pose the hazard of crystallization, precipitation, or degradation of some compounds, making it difficult to dispense accurate quantities in the future. Having samples stored in low-density plates requires a time consuming step of reformatting the samples into high-density plates before the high-density technology can be utilized.